Browsing by Author "Vaittinen, Anne"

RNA-sequencing is used to measure gene expression levels, characterize alternative splicing, identify SNPs and to study fusion genes. By using this information it is possible to understand function of different genetic elements and to understand embryogenesis as well as disease. The aim of this study was to compare new strand specific RNA sample preparation methods for next generation sequencing (NGS). The new method should have for example the following features: quick library preparation, smaller amount of starting material and strand specificity. Strand specificity is essential if there is no reference genome available or for example when the overlapping antisense transcripts are studied. Two different samples were prepared exactly according to each of four protocols that were tested. One of the samples was an RNA sample extracted from human blood and the other one was a BT474 cell line sample. The methods that were tested were NEXTflex Directional RNA-Seq Kit (Bioo Scientific), NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs), ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) and TotalScript RNA-Seq Kit (Epicentre). Laboratory’s non-strand specific method, Nextera (Illumina, New England BioLabs), which is custom modified to be applied for RNA, was used as a control method. Prepared RNA libraries were sequenced and the data compared for instance by using dendograms, strandedness and read coverage. After the comparison of the results and the usability of the methods it was found out that the ScriptSeq v2 RNA-Seq Library Preparation Kit was the most suitable of the methods for the laboratory’s usage.